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T-cell large granular lymphocytic (T-LGL) leukemia is a rare, typically indolent neoplasm with a median age of onset above 60 years. Pathogenesis involves clonal T-cell expansion, and nearly all reported pediatric cases have been associated with concurrent autoimmune disease. Immunosuppressive therapy often mitigates sequelae, but definitive cure is not routinely achieved. Here we present an otherwise healthy 13-year-old with T-LGL leukemia refractory to all standard treatments. Our patient ultimately underwent allogeneic bone marrow transplant (BMT) and is now stable in remission 3 years post-BMT. BMT may offer a viable definitive cure for refractory T-LGL leukemia in very young patients.

Prevention is a cornerstone of the guiding mission of the University of Virginia Comprehensive Cancer Center, which is “to reduce the burden of cancer for the patients of today, through skilled, integrated, and compassionate care and to eliminate the threat of cancer for the patients of tomorrow, through research and education in an environment that promotes diversity, equity, and inclusion.” We find it useful to conceptualize different opportunities for cancer prevention using NCI's Health Behaviors Research Branch's multilevel translational framework. The latter considers three intersecting continuums: cancer control—from prevention through survivorship; translation—from basic sciences to dissemination and implementation; and level of influence or impact—from genetics to policy. An advantage of this heuristic is that “prevention” is inherently defined as an inter-programmatic concept cutting across basic, clinical, and population science research rather than solely as a programmatic domain of Population Sciences. Through the UVA community outreach and engagement, we apply this multilevel framework to mitigate the social determinants of cancer risk and outcomes that drive cancer inequities in our catchment area. Below, we provide examples of our prevention research and translation along the model continuums and focus on equity.

Chronic natural killer large granular lymphocyte (NK-LGL) leukemia, also referred to as chronic lymphoproliferative disorder of NK cells, is a rare disorder defined by prolonged expansion of clonal NK cells. Similar prevalence of STAT3 mutations in chronic T-LGL and NK-LGL leukemia is suggestive of common pathogenesis. We undertook whole-genome sequencing to identify mutations unique to NK-LGL leukemia. The results were analyzed to develop a resequencing panel that was applied to 58 patients. Phosphatidylinositol 3-kinase pathway gene mutations (PIK3CD/PIK3AP1) and TNFAIP3 mutations were seen in 5% and 10% of patients, respectively. TET2 was exceptional in that mutations were present in 16 (28%) of 58 patient samples, with evidence that TET2 mutations can be dominant and exclusive to the NK compartment. Reduced-representation bisulfite sequencing revealed that methylation patterns were significantly altered in TET2 mutant samples. The promoter of TET2 and that of PTPRD, a negative regulator of STAT3, were found to be methylated in additional cohort samples, largely confined to the TET2 mutant group. Mutations in STAT3 were observed in 19 (33%) of 58 patient samples, 7 of which had concurrent TET2 mutations. Thrombocytopenia and resistance to immunosuppressive agents were uniquely observed in those patients with only TET2 mutation (Games-Howell post hoc test, P = .0074; Fisher’s exact test, P = .00466). Patients with STAT3 mutation, inclusive of those with TET2 comutation, had lower hematocrit, hemoglobin, and absolute neutrophil count compared with STAT3 wild-type patients (Welch’s t test, P ≤ .015). We present the discovery of TET2 mutations in chronic NK-LGL leukemia and evidence that it identifies a unique molecular subtype.

Classical MCL (cMCL) constitutes 6-8% of all B cell NHL. Despite recent advances, MCL is incurable except with allogeneic stem cell transplant. Blastic mantle cell lymphoma (bMCL) is a rarer subtype of cMCL associated with an aggressive clinical course and poor treatment response, frequent relapse and poor outcomes. We treated 13 bMCL patients with combined epigenetic and immunotherapy treatment consisting of vorinostat, cladribine and rituximab (SCR). We report an increased OS greater than 40 months with several patients maintaining durable remissions without relapse for longer than 5 years. This is remarkably better then current treatment regimens which in bMCL range from 14.5-24 months with conventional chemotherapy regimens. We demonstrate that the G/A870

Large granular lymphocyte (LGL) leukemia is a rare hematological disorder with expansion of the T‐cell or natural killer (NK) cell lineage. Signal transducer and activator of transcription 3 (STAT3) exhibits somatic activating mutations in 30%‐40% of LGL leukemia cases. Transcriptional targets of STAT3 include inflammatory cytokines, thus previous studies have measured cytokine levels of LGL leukemia patients compared to normal donors. Sphingolipid metabolism is a growing area of cancer research, with efforts focused on drug discovery. To date, no studies have examined serum sphingolipids in LGL leukemia patients, and only one study compared a subset of cytokines between the T‐LGL and NK‐LGL subtypes. Therefore, here, we included both LGL leukemia subtypes with the goals of (a) measuring serum sphingolipids for the first time, (b) measuring cytokines to find distinctions between the subtypes, and (c) establishing relationships with STAT3 mutations and clinical data. The serum analyses identified cytokines (EGF, IP‐10, G‐CSF) and sphingolipids (SMC22, SMC24, SMC20, LysoSM) significantly different in the LGL leukemia group compared to normal donors. In a mixed STAT3 mutation group, D661Y samples exhibited the highest mean corpuscular volume (MCV) values. We explored this further by expanding the cohort to include larger groups of single STAT3 mutations. Male D661Y STAT3 samples had lower Hgb and higher MCV compared to wild type (WT) or Y640F counterparts. This is the first report examining large groups of individual STAT3 mutations. Overall, our results revealed novel serum biomarkers and evidence that D661Y mutation may show different clinical manifestation compared to WT or Y640F STAT3., Analysis of LGL leukemia serum identified cytokines (EGF, IP‐10, G‐CSF) and sphingolipids (SMC22, SMC24, SMC20, LysoSM) that were significantly different compared to normal donors. Comparing three groups of STAT3 mutation status (WT, Y640F, D661Y) in LGL leukemia patients showed the D661Y mutation group had higher mean corpuscular volume and lower hemoglobin compared to the other groups.

Extranodal natural killer/T-cell lymphoma (ENKTL) is an aggressive, rare lymphoma of natural killer (NK) cell origin with poor clinical outcomes. Here we used phenotypic and molecular profiling, including epigenetic analyses, to investigate how ENKTL ontogeny relates to normal NK-cell development. We demonstrate that neoplastic NK cells are stably, but reversibly, arrested at earlier stages of NK-cell maturation. Genes downregulated in the most epigenetic immature tumors were associated with polycomb silencing along with genomic gain and overexpression of EZH2. ENKTL cells exhibited genome-wide DNA hypermethylation. Tumor-specific DNA methylation gains were associated with polycomb-marked regions, involving extensive gene silencing and loss of transcription factor binding. To investigate therapeutic targeting, we treated novel patient-derived xenograft (PDX) models of ENKTL with the DNA hypomethylating agent, 5-azacytidine. Treatment led to reexpression of NK-cell developmental genes, phenotypic NK-cell differentiation, and prolongation of survival. These studies lay the foundation for epigenetic-directed therapy in ENKTL. Significance: Through epigenetic and transcriptomic analyses of ENKTL, a rare, aggressive malignancy, along with normal NK-cell developmental intermediates, we identified that extreme DNA hypermethylation targets genes required for NK-cell development. Disrupting this epigenetic blockade in novel PDX models led to ENKTL differentiation and improved survival. This article is highlighted in the In This Issue feature, p. 85

Acute myeloid leukemia (AML) is an aggressive, heterogenous malignancy characterized by clonal expansion of bone marrow-derived myeloid progenitor cells. While our current understanding of the molecular and genomic landscape of AML has evolved dramatically and opened avenues for molecularly targeted therapeutics to improve upon standard intensive induction chemotherapy, curative treatments are elusive, particularly in older patients. Responses to current AML treatments are transient and incomplete, necessitating the development of novel treatment strategies to improve outcomes. To this end, harnessing the power of bioactive sphingolipids to treat cancer shows great promise. Sphingolipids are involved in many hallmarks of cancer of paramount importance in AML. Leukemic blast survival is influenced by cellular levels of ceramide, a bona fide pro-death molecule, and its conversion to signaling molecules such as sphingosine-1-phosphate and glycosphingolipids. Preclinical studies demonstrate the efficacy of therapeutics that target dysregulated sphingolipid metabolism as well as their combinatorial synergy with clinically-relevant therapeutics. Thus, increased understanding of sphingolipid dysregulation may be exploited to improve AML patient care and outcomes. This review summarizes the current knowledge of dysregulated sphingolipid metabolism in AML, evaluates how pro-survival sphingolipids promote AML pathogenesis, and discusses the therapeutic potential of targeting these dysregulated sphingolipid pathways.

Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. Vinca alkaloids such as vincristine (VCR), widely used in cancer treatment, are no exception, as their beneficial actions are often supplanted by resistance. In the present work we utilized HL-60 human leukemia cells and a VCR-resistant counterpart, HL-60/VCR, as a model to determine potential interplay between SL metabolism and mitochondrial bioenergetics supportive of multidrug resistance (MDR). Relative to wild-type cells, HL-60/VCR presented with global alterations in SL composition, typified by upregulated expression of sphingosine kinase (SPHK1), which catalyzes formation of sphingosine 1-phosphate (S1P), glucosylceramide synthase (GCS), which catalyzes formation of glucosylceramides (GC), and acid ceramidase, responsible for ceramide hydrolysis. In support of these changes, VCR resistance was also characterized by increases in S1P, several molecular species of ceramide and GC, and changes in sphingomyelin (SM) molecular species. With respect to mitochondria, despite increased basal respiration in HL-60/VCR cells, direct interrogation of the mitochondrial network revealed intrinsic respiratory complex insufficiency, largely localized to complex I (CI). Importantly, forced ceramide accumulation in wild-type cells phenocopied the respiratory insufficiency observed in HL-60/VCR, and co-targeting SL metabolism and CI induced synergistic cytotoxicity in HL-60/VCR cells, as well as in other MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.

We inquire how early in childhood children most at risk for problematic patterns of internalizing and externalizing behaviors can be accurately classified. Yearly measures of anxiety/depressive symptoms and aggressive behaviors (ages 6–13; n = 334), respectively, are used to identify behavioral trajectories. We then assess the degree to which limited spans of yearly information allow for the correct classification into the elevated, persistent pattern of the problem behavior, identified theoretically and empirically as high-risk and most in need of intervention. The true positive rate (sensitivity) is below 70% for anxiety/depressive symptoms and aggressive behaviors using behavioral information through ages 6 and 7. Conversely, by age 9, over 90% of the high-risk individuals are correctly classified (i.e., sensitivity) for anxiety/depressive symptoms, but this threshold is not met until age 12 for aggressive behaviors. Notably, the false positive rate of classification for both high-risk problem behaviors is consistently low using each limited age span of data (

Key Points Distinct sphingolipid metabolism of AML with MDS-related changes defines unique sensitivity to nanoliposomal C6-ceramide. Vinblastine alters sphingolipid metabolism to enhance the sensitivity of AML to nanoliposomal C6-ceramide.

Acute myelogenous leukemia (AML) is a hematological malignancy marked by the accumulation of large numbers of immature myeloblasts in bone marrow. The overall prognosis in AML is poor; hence, there is a pressing need to improve treatment. Although the sphingolipid (SL) ceramide demonstrates known cancer suppressor properties, it’s mechanism of action is multifaceted. Our studies in leukemia and other cancers have demonstrated that when combined with the antiestrogen, tamoxifen, the apoptosis-inducting effect of ceramide is greatly enhanced. The goal of the present study was to establish whether a ceramide-tamoxifen regimen also affects autophagic-driven cellular responses in leukemia. Using the human AML cell line KG-1, we demonstrate that, unlike exposure to the single agents, combination C6-ceramide-tamoxifen upregulated LC3-II expression, inhibited the mTOR signaling pathway, and synergistically induced KG-1 cell death in an Atg5-dependent manner. In addition, colocalization of autophagosome and mitochondria, indicative of mitophagosome formation and mitophagy, was observed. Versatility of the drug regimen was confirmed by experiments in MV4-11 cells, a FLT3-ITD AML mutant. These results indicate that the C6-ceramide-tamoxifen regimen plays a pivotal role inducing autophagy in AML, and thus constitutes a novel therapeutic design.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of older AML patients fail to respond to cytotoxic chemotherapy, and most responders relapse with drug-resistant disease. Failure to achieve complete remission can be partly attributed to the drug resistance advantage of AML blasts that frequently express P-glycoprotein (P-gp), an ATP-binding cassette transporter. Our previous work showed that elevated acid ceramidase (AC) levels in AML contribute to blast survival. Here, we investigated P-gp expression levels in AML relative to AC. Using parental HL-60 cells and drug-resistant derivatives as our model, we found that P-gp expression and efflux activity were highly upregulated in resistant derivatives. AC overexpression in HL-60 conferred resistance to the AML chemotherapeutic drugs, cytarabine, mitoxantrone, and daunorubicin, and was linked to P-gp upregulation. Furthermore, targeting AC through pharmacologic or genetic approaches decreased P-gp levels and increased sensitivity to chemotherapeutic drugs. Mechanistically, AC overexpression increased NF-κB activation whereas NF-kB inhibitors reduced P-gp levels, indicating that the NF-kappaB pathway contributes to AC-mediated modulation of P-gp expression. Hence, our data support an important role for AC in drug resistance as well as survival and suggest that sphingolipid targeting approaches may also impact drug resistance in AML.

T-LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of constitutive activation of the STAT3 and ERK pathway. Notably, in 40% of patients, constitutive STAT3 activation is due to STAT3 activating mutations, whereas in 60% this is unknown. As miRNAs are amongst the most potent regulators in health and disease, we hypothesized that aberrant miRNA expression could contribute to dysregulation of these pathways. miRNA sequencing in T-LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miR-181a. Furthermore, geneset enrichment analysis (GSEA) of downregulated targets of miR-181a implicated involvement in regulating STAT3 and ERK1/2 pathways. Flow cytometric analyses showed increased SOCS3+ and DUSP6+ T-LGL cells upon miR-181a inhibition. In addition, miR-181a-transfected human CD8+ T cells showed increased basal STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line, we could show that miR-181a is an actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition and ERK1/2 phosphorylation by DUSP6 inhibition and verified this mechanism in an independent cell line. In addition, miR-181a inhibition resulted in a higher sensitivity to FAS-mediated apoptosis. Collectively, our data show that miR-181a could be the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

PURPOSE OF REVIEW: The past decade in LGL leukemia research has seen increased pairing of clinical data with molecular markers, shedding new insights on LGL leukemia pathogenesis and heterogeneity. This review summarizes the current standard of care of LGL leukemia, updates from clinical trials, and our congruent improved understanding of LGL pathogenesis. RECENT FINDINGS: Various clinical reports have identified associations between stem, bone-marrow, and solid organ transplants and incidence of LGL leukemia. There is also a potential for underdiagnosis of LGL leukemia within the rheumatoid arthritis patient population, emphasizing our need for continued study. Preliminary results from the BNZ-1 clinical trial, which targets IL-15 along with IL-2 and IL-9 signaling pathways, show some evidence of clinical response. SUMMARY: With advances in our understanding of LGL pathogenesis from both the bench and the clinic, exciting avenues for investigations lie ahead for LGL leukemia.

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Acute myeloid leukemia (AML) is a disease characterized by uncontrolled proliferation of immature myeloid cells in the blood and bone marrow. The 5-year survival rate is approximately 25%, and recent therapeutic developments have yielded little survival benefit. Therefore, there is an urgent need to identify novel therapeutic targets. We previously demonstrated that acid ceramidase (ASAH1, referred to as AC) is upregulated in AML and high AC activity correlates with poor patient survival. Here, we characterized a novel AC inhibitor, SACLAC, that significantly reduced the viability of AML cells with an EC50 of approximately 3 μmol/L across 30 human AML cell lines. Treatment of AML cell lines with SACLAC effectively blocked AC activity and induced a decrease in sphingosine 1-phosphate and a 2.5-fold increase in total ceramide levels. Mechanistically, we showed that SACLAC treatment led to reduced levels of splicing factor SF3B1 and alternative MCL-1 mRNA splicing in multiple human AML cell lines. This increased proapoptotic MCL-1S levels and contributed to SACLAC-induced apoptosis in AML cells. The apoptotic effects of SACLAC were attenuated by SF3B1 or MCL-1 overexpression and by selective knockdown of MCL-1S. Furthermore, AC knockdown and exogenous C16-ceramide supplementation induced similar changes in SF3B1 level and MCL-1S/L ratio. Finally, we demonstrated that SACLAC treatment leads to a 37% to 75% reduction in leukemic burden in two human AML xenograft mouse models. IMPLICATIONS: These data further emphasize AC as a therapeutic target in AML and define SACLAC as a potent inhibitor to be further optimized for future clinical development., The authors thank those who generously provided cell lines for our studies: Dr. Jacqueline Cloos and Carolien van Alphen, VU Medical Center Amsterdam (EOL-1, HEL, Kasumi-3, Kasumi-6, ME-1, ML2, MM-6, and NB4); Dr. Mark Levis, Johns Hopkins Medical Institutions (MOLM-13 and MOLM-14); Dr. Douglas Graham, Emory University (NOMO1); Dr. Xiaorong Gu, Cleveland Clinic (OCI-AML2 and OCI-AML3); Dr. Harold L. Atkins, Ottawa Hospital Research Institute (OCI-AML4); Drs. Scott Kaufmann and Mithun Shah, Mayo Clinic (SET2); and Barbara Miller, Penn State Hershey (U937-Luc2-P2A-tdTomato). The authors also thank Alex Wendling, Wendy Dunton, Emily Sullins, Matthew Schmachtenberg, and Shubha Dighe (University of Virginia), and Viola Devine (Penn State, Hershey, PA) for technical assistance; Tye Deering (University of Virginia) for his lipid expertise; and Antonio Delgado (IQAC-CSIC) for SACLAC and RBM14C12 synthesis. The authors thank Marieke Jones (University of Virginia) for her statistics expertise. The authors thank the staff of the Flow Cytometry Core at Penn State University College of Medicine (Hershey, PA). The authors thank Drs. Melissa Jurica and Arun Ghosh (University of California, Santa Cruz, CA) for providing SSA for these studies. The authors also thank Drs. Samar Alsafadi and Marc-Henri Stern (Curie Institute) for providing the vector for SF3B1 overexpression. This work was supported by the NIH under the NCI Award Number P01CA171983 (to T.P. Loughran and M. Kester), P30CA044579 (to T.P. Loughran), and under the National Institute of General Medicine Sciences Award Number T32GM007055 (to J.M. Pearson) and AEI/FEDER (grant number CTQ2017-85378-R; to G. Fabrias). Additional funding was provided to T.P. Loughran by the Bess Family Charitable Fund and a generous anonymous donor. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Aggressive natural killer-cell (NK-cell) leukemia (ANKL) is an extremely aggressive malignancy with dismal prognosis and lack of targeted therapies. Here, we elucidate the molecular pathogenesis of ANKL using a combination of genomic and drug sensitivity profiling. We study 14 ANKL patients using whole-exome sequencing (WES) and identify mutations in STAT3 (21%) and RAS-MAPK pathway genes (21%) as well as in DDX3X (29%) and epigenetic modifiers (50%). Additional alterations include JAK-STAT copy gains and tyrosine phosphatase mutations, which we show recurrent also in extranodal NK/T-cell lymphoma, nasal type (NKTCL) through integration of public genomic data. Drug sensitivity profiling further demonstrates the role of the JAK-STAT pathway in the pathogenesis of NK-cell malignancies, identifying NK cells to be highly sensitive to JAK and BCL2 inhibition compared to other hematopoietic cell lineages. Our results provide insight into ANKL genetics and a framework for application of targeted therapies in NK-cell malignancies., Article, NATURE COMMUNICATIONS.9:1567(2018)

Acute myeloid leukemia (AML) is the most common adult leukemia. Only a fraction of AML patients will survive with existing chemotherapy regimens. Hence, there is an urgent and unmet need to identify novel targets and develop better therapeutics in AML. In the past decade, the field of sphingolipid metabolism has emerged into the forefront of cancer biology due to its importance in cancer cell proliferation and survival. In particular, acid ceramidase (AC) has emerged as a promising therapeutic target due to its role in neutralizing the pro-death effects of ceramide. Areas covered: This review highlights key information about AML biology as well as current knowledge on dysregulated sphingolipid metabolism in cancer and AML. We describe AC function and dysregulation in cancer, followed by a review of studies that report elevated AC in AML and compounds known to inhibit the enzyme. Expert opinion: AML has a great need for new drug targets and better therapeutic agents. The finding of elevated AC in AML supports the concept that this enzyme represents a novel and realistic therapeutic target for this common leukemia. More effort is needed towards developing better AC inhibitors for clinical use and combination treatment with existing AML therapies.

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The combination of daunorubicin (dnr) and cytarabine (Ara-C) is a cornerstone of treatment for acute myelogenous leukemia (AML); resistance to these drugs is a major cause of treatment failure. Ceramide, a sphingolipid (SL), plays a critical role in cancer cell apoptosis in response to chemotherapy. Here, we investigated the effects of chemotherapy selection pressure with Ara-C and dnr on SL composition and enzyme activity in the AML cell line HL-60. Resistant cells, those selected for growth in Ara-C- and dnr-containing medium (HL-60/Ara-C and HL-60/dnr, respectively), demonstrated upregulated expression and activity of glucosylceramide synthase, acid ceramidase (AC), and sphingosine kinase 1 (SPHK1); were more resistant to ceramide than parental cells; and displayed sensitivity to inhibitors of SL metabolism. Lipidomic analysis revealed a general ceramide deficit and a profound upswing in levels of sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) in HL-60/dnr cells versus parental and HL-60/Ara-C cells. Both chemotherapyselected cells also exhibited comprehensive upregulations in mitochondrial biogenesis consistent with heightened reliance on oxidative phosphorylation, a property that was partially reversed by exposure to AC and SPHK1 inhibitors and that supports a role for the phosphorylation system in resistance. In summary, dnr and Ara-C selection pressure induces acute reductions in ceramide levels and large increases in S1P and C1P, concomitant with cell resilience bolstered by enhanced mitochondrial remodeling. Thus, strategic control of ceramide metabolism and further research to define mitochondrial perturbations that accompany the drug-resistant phenotype offer new opportunities for developing therapies that regulate cancer growth., This work was supported by National Institutes of Health Grant P01 CA171983 and by a grant from the Brody Brothers Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declare that they have no conflicts of interest with the contents of this article.

Genomic analysis of cancer offers the hope of identifying new treatments or aiding in the selection of existing treatments. Rare leukemias pose additional challenges in this regard as samples may be hard to acquire and when found the underlying pathway may not be attractive to drug development since so few individuals are affected. In this case, it can be useful to identify common mutational overlap among subsets of rare leukemias to increase the number of individuals that may benefit from a targeted therapy. This chapter examines the current mutational landscape of large granular lymphocyte (LGL) leukemia with a focus on STAT3 mutations, the most common mutation in LGL leukemia to date. We examined the linkage between these mutations and autoimmune symptoms and disorders, in cases of obvious and suspected LGL leukemia. We then summarized and compared mutations in a set of other rare leukemias that also have JAK/STAT signaling pathway activation brought about by genomic changes. These include T-cell acute lymphoblastic leukemia (T-ALL), T-cell prolymphocytic leukemia (T-PLL), cutaneous T-cell lymphoma (CTCL), select peripheral T-cell lymphoma (PTCL), and adult T-cell leukemia/lymphoma (ATLL). Though STAT3 activation is common in these leukemias, the way in which it is achieved, such as the activating cytokine pathway and/or the co-mutational background, is quite diverse.

The growing public health and legal concerns regarding gun violence has led to a call for research that investigates risk factors for gun violence across a variety of domains. Individual and sociocontextual risk factors have been associated with violence more broadly, and in some instances gun-carrying, however no prior research has investigated the role of these factors in explaining gun violence using longitudinal data. The current study utilized a subsample (N = 161) from the Pathways to Desistance Study, which is a longitudinal sample of serious adolescent offenders to evaluate interindividual and intraindividual differences in relevant risk factors of gun violence. Results suggest that there are a few key proximal individual-level and sociocontextual predictors for gun violence, including witnessing nongun violence, future orientation, and perceived personal rewards to crime. Findings demonstrate the salience of exposure to violence in contributing to gun violence and identify levers of action for public policy. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

BackgroundLarge granular lymphocyte (LGL) leukemia is an uncommon cancer characterized by a sustained clonal proliferation of LGL cells. Antibodies reactive to retroviruses have been documented in the serum of patients with LGL leukemia. Culture or molecular approaches have to date not been successful in identifying a retrovirus.MethodsBecause a retrovirus must integrate into the genome of an infected cell, we focused our efforts on detecting a novel retrovirus integration site in the clonally expanded LGL cells. We present a new computational tool that uses long-insert mate pair sequence data to search the genome of LGL leukemia cells for retrovirus integration sites. We also utilize recently published methods to interrogate the status of polymorphic human endogenous retrovirus type K (HERV-K) provirus in patient genomes.ResultsWhile our analysis did not reveal any new retrovirus insertions in LGL genomes from LGL leukemia patients, we did identify four HERV-K provirus integration sites that are polymorphic in the human population and absent from the human reference genome, hg19. To determine if the prevalence of these or other polymorphic proviral HERV-Ks differed between LGL leukemia patients and the general population, we applied a recently developed approach that reports all sites in the human genome occupied by a proviral HERV-K. Using the 1000 genomes project (KGP) data as a reference database for HERV-K proviral prevalence at each polymorphic site, we show that there are significant differences in the number of polymorphic HERV-Ks in the genomes of LGL leukemia patients of European origin compared to individuals with European ancestry in the KGP data.ConclusionsOur study confirms that the integration of a new infectious or endogenous retrovirus does not cause the clonal expansion of LGL cells in LGL leukemia, although we do not rule out that these cells could be responding to retroviral antigens produced in other cell types. However, it is of interest that the burden of polymorphic proviral HERV-K is elevated in LGL leukemia patient genomes. Our research emphasizes the merits of comprehensive genomic assessment of HERV-K in cancer samples and suggests that further analyses to determine contributions of HERV-K to LGL leukemia are warranted.

Commonly known for its critical role in calcium homeostasis and bone mineralization, more recently vitamin D has been implicated in haematological cancer pathogenesis and shows promise as an anti-cancer therapy. Serum levels of 25(OH)D3, the precursor to the active form of vitamin D, calcitriol, are typically lower in patients with haematological disease compared to healthy individuals. This often correlates with worse disease outcome. Furthermore, diseased cells typically highly express the vitamin D receptor (VDR), which is required for many of the anti-cancer effects observed in multiple in vivo and in vitro cancer models. In abnormal haematological cells, vitamin D supplementation promotes apoptosis, induces differentiation, inhibits proliferation, sensitizes tumor cells to other anti-cancer therapies, and reduces the production of pro-inflammatory cytokines. Although the dosage of vitamin D required to achieve these effects may induce hypercalcemia in humans, analogs and combinatorial treatments have been developed to circumvent this side effect. Vitamin D and its analogs are well tolerated in clinical trials and thus further investigation into the use of these agents in the clinic is warranted. Here we review the current literature in this field.

Large granular lymphocyte leukemia (LGLL) is a rare incurable chronic disease typically characterized by clonal expansion of CD3+ cytotoxic T-cells. Two signal transducer and activator of transcription factors, STAT1 and STAT3, are constitutively active in T-LGLL. Disruption of this activation induces apoptosis in T-LGLL cells. Therefore, considerable efforts are focused on developing treatments that inhibit STAT activation. Calcitriol, the active form of vitamin D, has been shown to decrease STAT1 and STAT3 phosphorylation in cancer cell lines and autoimmune disease mouse models. Thus, we investigated whether calcitriol could be a valid therapeutic for T-LGLL. Calcitriol treatment of the TL-1 cell line (model of T-LGLL) led to decreased phospho-Y701 STAT1 and phospho-Y705 STAT3 and increased vitamin D receptor (VDR) levels. Doses of 10 and 100 nM calcitriol also significantly decreased the inflammatory cytokine IFN-γ in the TL-1 cell line. The overall cell viability did not change when the TL-1 cell line was treated with 0.1 to 1000 nM calcitriol. Studies with primary T-LGLL patient peripheral blood mononuclear cells showed that the majority of T-LGLL patients have detectable VDR and activated STATs in contrast to normal donor controls. Treatment of primary T-LGLL patient cells with calcitriol recapitulated findings from the TL-1 cell line. Overall, our results suggest that calcitriol may reprogram T-cells to decrease essential STAT activation and pro-inflammatory cytokine output. These data support further investigation into calcitriol as an experimental therapeutic for T-LGLL.

Donor-derived CD4(+) T lymphocytes are the major effector cells directly involved in the development of graft-versus-host disease (GVHD). As a negative regulator of immune cell differentiation and development, microRNA-150 (miR-150) induces immunological tolerance in CD4(+) T cells after transplantation. However, the specific mechanisms have not been fully elucidated. In this study, we demonstrated that miR-150 is capable of not only inhibiting proliferation and activation of CD4(+) T cells but also promoting apoptosis. Mechanistically, miR-150 targets v-akt murine thymoma viral oncogene homolog 3 (AKT3), and subsequently downregulates B-cell lymphoma 2 (Bcl-2) interacting mediator of cell death (BIM). We have also demonstrated that re-expression of AKT3 reversed miR-150-mediated inhibition of CD4(+) T lymphocyte development. Therefore, we conclude that miR-150 negatively regulates CD4(+) T cell function by inhibiting the AKT3/BIM signaling pathway. These findings also suggest that manipulating the levels of miRNA-150 could be a valuable strategy in prevention and/or treatment of acute graft-versus-host disease.

Calcitriol, the active form of vitamin D, has been well documented to act directly on immune cells and malignant cells. Activated T cells are one of the best characterized targets of calcitriol, with effects including decreasing inflammatory cytokine output and promoting anti-inflammatory cytokine production. However, the effects of calcitriol on natural killer (NK) cells are less clear. Reports suggest that only immature NK cell populations are affected by calcitriol treatment resulting in impaired cytotoxic function and cytokine production, while mature NK cells may have little or no response. NK cell large granular lymphocyte leukemia (NK-LGLL) is a rare leukemia with CD3-CD16+CD56+NK cell clonal expansion. The current standard treatments are immunosuppressant therapies, which are not curative. The Janus kinase (JAK) – signal transducer and activator of transcription (STAT) pathway is hyperactivated in LGLL and is one pathway of interest in new drug target investigations. We previously demonstrated the ability of calcitriol to decrease STAT1 tyrosine 701 (p-STAT1) and STAT3 tyrosine 705 (p-STAT3) phosphorylation as well as inflammatory cytokine output of T cell large granular lymphocyte leukemia cells, but did not determine the effects of calcitriol on NK-LGLL. Therefore, in the present study, we investigated whether NKL cells, a model of NK-LGLL, and NK-LGLL patient peripheral blood mononuclear cells (PBMCs) are susceptible to treatment with calcitriol or seocalcitol (EB1089), a potent analog of calcitriol. NKL cells are dependent on interleukin (IL)-2 for survival and we show here for the first time that treatment with IL-2 induced tyrosine phosphorylation of STATs 1 through 6. Both calcitriol and EB1089 caused significant upregulation of the vitamin D receptor (VDR). IL-2 induction of p-STAT1 and p-STAT3 phosphorylation was significantly decreased after calcitriol or EB1089 treatment. Additionally, IL-10, interferon (IFN)-γ, and FMS-like tyrosine kinase 3 ligand (Flt-3L) extracellular output was significantly decreased at 100 nM EB1089 and intracellular IL-10 was decreased with either calcitriol or EB1089 treatment. We treated NK-LGLL patient PBMCs with calcitriol or EB1089 and found decreased p-STAT1 and p-STAT3 while VDR increased, which matched the NKL cell line data. We then measured 75 serum cytokines in NK-LGLL patients (n = 8) vs. age- and sex-matched normal healthy donors (n = 8), which is the first serum cytokine study for this LGLL subtype. We identified 15 cytokines, including IL-10 and Flt-3L, which were significantly different between normal donors and NK-LGLL patients. Overall, our results suggest that activating the vitamin D pathway could be a mechanism to decrease STAT1 and 3 activation and inflammatory cytokine output in NK-LGLL patients.

Large granular lymphocyte (LGL) leukemia results from clonal expansion of CD3(+) cytotoxic T lymphocytes or CD3(−) natural killer (NK) cells. Chronic antigen stimulation is postulated to promote long-term survival of LGL leukemia cells through constitutive activation of multiple survival pathways, resulting in global dysregulation of apoptosis and resistance to activation-induced cell death. We reported previously that nuclear factor κB (NF-κB) is a central regulator of the survival network for leukemic LGL. However, the mechanisms that trigger constitutive activation of NF-κB in LGL leukemia remain undefined. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis in tumor cells but can also activate NF-κB through interaction with TRAIL receptors 1, 2, and 4 (also known as DR4, DR5, and DcR2, respectively). The role of TRAIL has not been studied in LGL leukemia. In this study, we hypothesized that TRAIL interaction with DcR2 contributes to NF-κB activation in LGL leukemia. We observed upregulated TRAIL messenger RNA and protein expression in LGL leukemia cells with elevated levels of soluble TRAIL protein in LGL leukemia patient sera. We also found that DcR2 is the predominant TRAIL receptor in LGL leukemia cells. We demonstrated that TRAIL-induced activation of DcR2 led to increased NF-κB activation in leukemic LGL. Conversely, interruption of TRAIL-DcR2 signaling led to decreased NF-κB activation. Finally, a potential therapeutic application of proteasome inhibitors (bortezomib and ixazomib), which are known to inhibit NF-κB, was identified through their ability to decrease proliferation and increase apoptosis in LGL leukemia cell lines and primary patient cells.

Interleukin-15 (IL-15) and IL-2 drive T-cell malignancies including T-cell large granular lymphocyte leukemia (T-LGLL) and HTLV-1 driven adult T-cell leukemia (ATL). Both cytokines share common γ-chain receptors and downstream signaling pathways. T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with abnormal JAK/STAT signaling. ATL is an aggressive CD4+ T-cell neoplasm associated with HTLV-1. T-LGLL and ATL share dependence on IL-2 and IL-15 for survival and both diseases lack effective therapies. BNZ-1 is a pegylated peptide designed to specifically bind the γc receptor to selectively block IL-2, IL-15, and IL-9 signaling. We hypothesized that treatment with BNZ-1 would reduce cytokine-mediated proliferation and viability. Our results demonstrated that in vitro treatment of a T-LGLL cell line and ex vivo treatment of T-LGLL patient cells with BNZ-1 inhibited cytokine-mediated viability. Furthermore, BNZ-1 blocked downstream signaling and increased apoptosis. These results were mirrored in an ATL cell line and in ex vivo ATL patient cells. Lastly, BNZ-1 drastically reduced leukemic burden in an IL-15-driven human ATL mouse xenograft model. Thus, BNZ-1 shows great promise as a novel therapy for T-LGLL, ATL, and other IL-2 or IL-15 driven hematopoietic malignancies.

T cell large granular lymphocyte leukemia (T-LGLL) is a rare incurable disease that is characterized by defective apoptosis of cytotoxic CD8+ T cells. Chronic activation of the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway is a hallmark of T-LGLL. One manifestation is the constitutive phosphorylation of tyrosine 701 of STAT1 (p-STAT1). T-LGLL patients also exhibit elevated serum levels of the STAT1 activator, interferon-γ (IFN-γ), thus contributing to an inflammatory environment. In normal cells, IFN-γ production is tightly controlled through induction of IFN-γ negative regulators. However, in T-LGLL, IFN-γ signaling lacks this negative feedback mechanism as evidenced by excessive IFN-γ production and decreased levels of suppressors of cytokine signaling 1 (SOCS1), a negative regulator of IFN-γ. Here we characterize the IFN-γ-STAT1 pathway in TL-1 cells, a cell line model of T-LGLL. TL-1 cells exhibited lower IFN-γ receptor protein and mRNA expression compared to an IFN-γ responsive cell line. Furthermore, IFN-γ treatment did not induce JAK2 or STAT1 activation or transcription of IFN-γ-inducible gene targets. However, IFN-β induced p-STAT1 and subsequent STAT1 gene transcription, demonstrating a specific IFN-γ signaling defect in TL-1 cells. We utilized siRNA targeting of STAT1, STAT3, and STAT5b to probe their role in IL-2-mediated IFN-γ regulation. These studies identified STAT5b as a positive regulator of IFN-γ production. We also characterized the relationship between STAT1, STAT3, and STAT5b proteins. Surprisingly, p-STAT1 was positively correlated with STAT3 levels while STAT5b suppressed the activation of both STAT1 and STAT3. Taken together, these results suggest that the dysregulation of the IFN-γ-STAT1 signaling pathway in TL-1 cells likely results from low levels of the IFN-γ receptor. The resulting inability to induce negative feedback regulators explains the observed elevated IL-2 driven IFN-γ production. Future work will elucidate the best way to target this pathway, with the ultimate goal to find a better therapeutic for T-LGLL.

Felty syndrome is a rare disease defined by neutropenia, splenomegaly, and rheumatoid arthritis. Sometimes the differential diagnosis between Felty syndrome and large granular lymphocyte leukemia is problematic. Recently, somatic STAT3 and STAT5B mutations were discovered in 30-40% of patients with large granular lymphocyte leukemia. Herein, we aimed to study whether these mutations can also be detected in Felty syndrome, which would imply the existence of a common pathogenic mechanism between these two disease entities. We collected samples and clinical information from 14 Felty syndrome patients who were monitored at the rheumatology outpatient clinic for Felty syndrome. Somatic STAT3 mutations were discovered in 43% (6/14) of Felty syndrome patients with deep amplicon sequencing targeting all STAT3 exons. Mutations were located in the SH2 domain of STAT3, which is a known mutational hotspot. No STAT5B mutations were found. In blood smears, overrepresentation of large granular lymphocytes was observed, and in the majority of cases the CD8(+) T-cell receptor repertoire was skewed when analyzed by flow cytometry. In bone marrow biopsies, an increased amount of phospho-STAT3 positive cells was discovered. Plasma cytokine profiling showed that ten of the 92 assayed cytokines were elevated both in Felty syndrome and large granular lymphocyte leukemia, and three of these cytokines were also increased in patients with uncomplicated rheumatoid arthritis. In conclusion, somatic STAT3 mutations and STAT3 activation are as frequent in Felty syndrome as they are in large granular lymphocyte leukemia. Considering that the symptoms and treatment modalities are also similar, a unified reclassification of these two syndromes is warranted.

Sphingolipid metabolism has been identified as a potential therapeutic target in cancer. Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid metabolite produced by sphingosine kinases-1 and -2 (SPHK1 and SPHK2). Elevated SPHK1 has been found in numerous cancer types and been shown to contribute to survival, chemotherapeutic resistance and malignancy. However, its role in large granular Natural Killer (NK) large granular lymphocyte (LGL) leukemia has not been investigated. Here, we examine SPHK1 as a therapeutic target in LGL leukemia. We found that SPHK1 is overexpressed in peripheral blood mononuclear cells (PBMCs) from LGL leukemia patients which results in elevated S1P in the sera. The use of SPHK1 inhibitors, SKI-II or SKI-178, decreased leukemic NK cell viability and induced caspase-dependent apoptosis. SKI-II and SKI-178 restored the sphingolipid balance by increasing ceramide and decreasing S1P in leukemic NKL cells. SKI-II and SKI-178 also induced apoptosis in primary NK-LGLs from leukemia patients. Mechanistic studies in NK-LGL cell lines demonstrated that SKI-178 and SKI-II induced cell cycle arrest at G2/M. We found that SKI-178 induced phosphorylation of Bcl-2 at Ser70, and that this was dependent on CDK1. We further show that SPHK1 inhibition with SKI-178 leads to decreased JAK-STAT signaling. Our data demonstrate that SPHK1 represents a novel therapeutic target for the treatment of NK-LGL leukemia.

The triphenylethylene antiestrogen, tamoxifen, can be an effective inhibitor of sphingolipid metabolism. This off-target activity makes tamoxifen an interesting ancillary for boosting the apoptosis-inducing properties of ceramide, a sphingolipid with valuable tumor censoring activity. Here we show for the first time that tamoxifen and metabolite, N-desmethyltamoxifen (DMT), block ceramide glycosylation and inhibit ceramide hydrolysis (by acid ceramidase, AC) in human acute myelogenous leukemia (AML) cell lines and in AML cells derived from patients. Tamoxifen (1–10 μM) inhibition of AC in AML cells was accompanied by decreases in AC protein expression. Tamoxifen also depressed expression and activity of sphingosine kinase 1 (SphK1), the enzyme-catalyzing production of mitogenic sphingosine 1-phosphate (S1-P). Results from mass spectroscopy showed that tamoxifen and DMT (i) increased the levels of endogenous C16:0 and C24:1 ceramide molecular species, (ii) nearly totally halted production of respective glucosylceramide (GC) molecular species, (iii) drastically reduced levels of sphingosine (to 9% of control), and (iv) reduced levels of S1-P by 85%, in vincristine-resistant HL-60/VCR cells. The co-administration of tamoxifen with either N-(4-hydroxyphenyl)retinamide (4-HPR), a ceramide-generating retinoid, or a cell-deliverable form of ceramide, C6-ceramide, resulted in marked decreases in HL-60/VCR cell viability that far exceeded single agent potency. Combination treatments resulted in synergistic apoptotic cell death as gauged by increased Annexin V binding and DNA fragmentation and activation of caspase-3. These results show the versatility of adjuvant triphenylethylene with ceramide-centric therapies for magnifying therapeutic potential in AML. Such drug regimens could serve as effective strategies, even in the multidrug-resistant setting.

Previously, we had shown that although only 8% of patients with large granular lymphocytic leukemia (LGLL) were infected with human T cell lymphoma/leukemia virus (HTLV)-2, almost half had antibodies to HTLV Gag and Env peptides. Herein, we investigated whether this could be due to cross-reactive antibodies to two homologous peptides in the Gag protein of the endogenous retrovirus HTLV-related endogenous sequence-1 (HRES-1). In addition, we had previously shown that patients with HTLV neurodegenerative diseases had increased seroreactivity to homologous HERV-K10 endogenous retrovirus peptides. Hence, in this study we also examined whether these patients had increased seroreactivity to the aforementioned HRES-1 Gag peptides. Sera from 100 volunteer blood donors (VBD), 53 patients with LGLL, 74 subjects with HTLV-1 or 2 infection (58 nonmyelopathy and 16 myelopathy), and 83 patients with multiple sclerosis (MS) were evaluated. The HTLV-positive myelopathy (HAM) patients had a statistically increased prevalence of antibodies to both HRES-1 Gag peptides (81%) vs. the VBD (0%), LGLL patients (13%), and MS patients (1%), and the HTLV-positive nonmyelopathy subjects (21%). The data suggest that cross-reactivity to HRES-1 peptides could be involved in the pathogenesis of HAM. The difference between the VBD and LGLL patients was also statistically significant, also suggesting a possible association in a minority of patients.

We previously developed SKI-178 (N′-[(1E)-1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178–induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178–induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178–induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the prosurvival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multidrug resistance mediated by multidrug-resistant protein1 and/or prosurvival Bcl-2 family member overexpression did not affect the sensitivity of AML cells to SKI-178. Taken together, these findings highlight the therapeutic potential of SKI-178 targeting SphK1 as a novel therapeutic agent for the treatment of AML, including multidrug-resistant/recurrent AML subtypes.

Objectives. We observed how perceptions of risks, costs, crime rewards, and violence exposure change as individual gun-carrying behavior changes among high-risk adolescents. Methods. We analyzed a longitudinal study (2000–2010) of serious juvenile offenders in Maricopa County, Arizona, or Philadelphia County, Pennsylvania, assessing within-person changes in risk and reward perceptions, and violence exposure as individuals initiated or ceased gun carrying. Results. Despite being associated with heightened exposure to violence, gun carrying was linked to lower perceptions of risks and costs and higher perceived rewards of offending. Gun carrying was not time-stable, as certain individuals both started and stopped carrying during the study. Within-person changes in carrying guns were associated with shifting perceptions of risks, costs, and rewards of crime, and changes in exposure to violence in expected directions. Conclusions. Gun carrying reduces perceptions of risks associated with offending while increasing actual risk of violence exposure. This suggests that there is an important disconnect between perceptions and objective levels of safety among high-risk youths. Gun-carrying decisions may not only be influenced by factors of protection and self-defense, but also by perceptions of risks and reward associated with engaging in crime more generally.

This section reviews the diagnostic criteria and pathogenesis of large granular lymphocyte (LGL) leukemia. There is a particular focus on the overlap of LGL leukemia and rheumatoid arthritis (Felty's syndrome). Current understanding of the mechanisms of neutropenia in these disorders is discussed. Finally, treatment indications and therapeutic recommendations are outlined.

Constitutively activated STAT1 and elevated IFN-γ are both characteristic of T cell large granular lymphocytic leukemia (T-LGLL), a rare incurable leukemia with clonal expansion of cytotoxic T cells due to defective apoptosis. Interferon gamma (IFN-γ) is an inflammatory cytokine that correlates with worse progression and symptomology in multiple autoimmune diseases and cancers. In canonical IFN-γ-STAT1 signaling, IFN-γ activates STAT1, a transcription factor, via phosphorylation of tyrosine residue 701 (p-STAT1). p-STAT1 then promotes transcription of IFN-γ, creating a positive feedback loop. We previously found that calcitriol treatment of the TL-1 cell line, a model of T-LGLL, significantly decreased IFN-γ secretion and p-STAT1 while increasing the vitamin D receptor (VDR) protein. Here we further explore these observations. Using TL-1 cells, IFN-γ decreased starting at 4 h following calcitriol treatment, with a reduction in the intracellular and secreted protein levels as well as the mRNA content. A similar reduction in IFN-γ transcript levels was observed in primary T-LGLL patient peripheral blood mononuclear cells (PBMCs). p-STAT1 inhibition followed a similar temporal pattern and VDR upregulation inversely correlated with IFN-γ levels. Using EB1089 and 25(OH)D(3), which have high or low affinity for VDR, respectively, we found that the decrease in IFN-γ correlated with the ability of EB1089, but not 25(OH)D(3), to upregulate VDR. However, both compounds inhibited p-STAT1; thus the reduction of p-STAT1 is not solely responsible for IFN-γ inhibition. Conversely, cells treated with VDR siRNA exhibited decreased basal IFN-γ production upon VDR knockdown in a dose-dependent manner. Calcitriol treatment upregulated VDR and decreased IFN-γ regardless of initial VDR knockdown efficiency, strengthening the connection between VDR upregulation and IFN-γ reduction. Our findings suggest multiple opportunities to further explore the clinical relevance of the vitamin D pathway and the potential role for vitamin D supplementation in T-LGLL.

The anticancer properties of ceramide, a sphingolipid with potent tumor-suppressor properties, can be dampened via glycosylation, notably in multidrug resistance wherein ceramide glycosylation is characteristically elevated. Earlier works using the ceramide analog, C6-ceramide, demonstrated that the antiestrogen tamoxifen, a first generation P-glycoprotein (P-gp) inhibitor, blocked C6-ceramide glycosylation and magnified apoptotic responses. The present investigation was undertaken with the goal of discovering non-anti-estrogenic alternatives to tamoxifen that could be employed as adjuvants for improving the efficacy of ceramide-centric therapeutics in treatment of cancer. Herein we demonstrate that the tamoxifen metabolites, desmethyltamoxifen and didesmethyltamoxifen, and specific, high-affinity P-gp inhibitors, tariquidar and zosuquidar, synergistically enhanced C6-ceramide cytotoxicity in multidrug resistant HL-60/VCR acute myelogenous leukemia (AML) cells, whereas the selective estrogen receptor antagonist, fulvestrant, was ineffective. Active C6-ceramide-adjuvant combinations elicited mitochondrial ROS production and cytochrome c release, and induced apoptosis. Cytotoxicity was mitigated by introduction of antioxidant. Effective adjuvants markedly inhibited C6-ceramide glycosylation as well as conversion to sphingomyelin. Active regimens were also effective in KG-1a cells, a leukemia stem cell-like line, and in LoVo human colorectal cancer cells, a solid tumor model. In summary, our work details discovery of the link between P-gp inhibitors and the regulation and potentiation of ceramide metabolism in a pro-apoptotic direction in cancer cells. Given the active properties of these adjuvants in synergizing with C6-ceramide, independent of drug resistance status, stemness, or cancer type, our results suggest that the C6-ceramide-containing regimens could provide alternative, promising therapeutic direction, in addition to finding novel, off-label applications for P-gp inhibitors.

A facultative heterochromatin mark, histone H3 lysine 9 dimethylation (H3K9me2), which is mediated by histone methyltransferases G9a/GLP (EHMT2/1), undergoes dramatic rearrangements during myeloid cell differentiation as observed by chromatin imaging. To determine whether these structural transitions also involve genomic repositioning of H3K9me2, we used ChIP-sequencing to map genome-wide topography of H3K9me2 in normal human granulocytes, normal CD34+ hematopoietic progenitors, primary myeloblasts from acute myeloid leukemia (AML) patients, and a model leukemia cell line K562. We observe that H3K9me2 naturally repositions from the previously designated “repressed” chromatin state in hematopoietic progenitors to predominant association with heterochromatin regions in granulocytes. In contrast, AML cells accumulate H3K9me2 on previously undefined large (> 100 Kb) genomic blocks that are enriched with AML-specific single nucleotide variants, sites of chromosomal translocations, and genes downregulated in AML. Specifically, the AML-specific H3K9me2 blocks are enriched with genes regulated by the proto-oncogene ERG that promotes stem cell characteristics. The AML-enriched H3K9me2 blocks (in contrast to the heterochromatin-associated H3K9me2 blocks enriched in granulocytes) are reduced by pharmacological inhibition of the histone methyltransferase G9a/GLP in K562 cells concomitantly with transcriptional activation of ERG and ETS1 oncogenes. Our data suggest that G9a/GLP mediate formation of transient H3K9me2 blocks that are preserved in AML myeloblasts and may lead to an increased rate of AML-specific mutagenesis and chromosomal translocations.

Large granular lymphocyte (LGL) leukemia has been recognized by the World Health Organization classifications amongst mature T-cell and natural killer (NK) cell neoplasms. There are 3 categories: chronic T-cell leukemia and NK-cell lymphocytosis, which are similarly indolent diseases characterized by cytopenias and autoimmune conditions as opposed to aggressive NK-cell LGL leukemia. Clonal LGL expansion arise from chronic antigenic stimulation, which promotes dysregulation of apoptosis, mainly due to constitutive activation of survival pathways including Jak/Stat, MapK, phosphatidylinositol 3-kinase–Akt, Ras–Raf-1, MEK1/extracellular signal-regulated kinase, sphingolipid, and nuclear factor-κB. Socs3 downregulation may also contribute to Stat3 activation. Interleukin 15 plays a key role in activation of leukemic LGL. Several somatic mutations including Stat3, Stat5b, and tumor necrosis factor alpha-induced protein 3 have been demonstrated recently in LGL leukemia. Because these mutations are present in less than half of the patients, they cannot completely explain LGL leukemogenesis. A better mechanistic understanding of leukemic LGL survival will allow future consideration of a more targeted therapeutic approach than the current practice of immunosuppressive therapy.

Mcl-1, a pro-survival member of the Bcl-2 protein family, is an attractive target for cancer therapy. We have recently identified the natural product marinopyrrole A (maritoclax) as a novel small molecule Mcl-1 inhibitor. Here, we describe the structure-activity relationship study of pyoluteorin derivatives based on maritoclax. To date, we synthesized over 30 derivatives of maritoclax and evaluated their inhibitory actions and cytotoxicity toward Mcl-1-dependent cell lines. As a result, several functional groups were identified in the pyoluteorin motif that significantly potentiate biological activity. A number of such derivatives, KS04 and KS18, interacted with Mcl-1 in a conserved fashion according to NMR spectroscopy and molecular modeling. KS04 and KS18 induced apoptosis selectively in Mcl-1-dependent but not Bcl-2-dependent K562 cells through selective Mcl-1 down-regulation, and synergistically enhanced apoptosis in combination with ABT-737. Moreover, the intraperitoneal administration of KS18 (10 mg/kg/d) and ABT-737 (20 mg/kg/d) significantly suppressed the growth of ABT-737-resistant HL-60 xenografts in nude mice without apparent toxicity. Overall, we identified the pharmacophore of pyoluteorin derivatives that act as potent and promising Mcl-1 antagonists against Mcl-1-dependent hematological cancers.

Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to leave the primary tumor site, invade surrounding tissue, and establish distant metastases. A hallmark of EMT is the loss of E-cadherin expression, and one major signal for the induction of EMT is TGFβ, which is dysregulated in up to 40% of hepatocellular carcinoma (HCC). We have constructed an EMT network of 70 nodes and 135 edges by integrating the signaling pathways involved in developmental EMT and known dysregulations in invasive HCC. We then used discrete dynamic modeling to understand the dynamics of the EMT network driven by TGFβ. Our network model recapitulates known dysregulations during the induction of EMT and predicts the activation of the Wnt and Sonic hedgehog (SHH) signaling pathways during this process. We show, across multiple murine (P2E and P2M) and human HCC cell lines (Huh7, PLC/PRF/5, HLE, and HLF), that the TGFβ signaling axis is a conserved driver of mesenchymal phenotype HCC and confirm that Wnt and SHH signaling are induced in these cell lines. Furthermore, we identify by network analysis eight regulatory feedback motifs that stabilize the EMT process and show that these motifs involve cross-talk among multiple major pathways. Our model will be useful in identifying potential therapeutic targets for the suppression of EMT, invasion, and metastasis in HCC. Cancer Res; 74(21); 5963–77. ©2014 AACR.

T-cell large granular lymphocytic leukemia and chronic lymphoproliferative disorder of natural killer cells are intriguing entities between benign and malignant lymphoproliferation. The molecular pathogenesis has partly been uncovered by the recent discovery of somatic activating STAT3 and STAT5b mutations. Here we show that 43% (75/174) of patients with T-cell large granular lymphocytic leukemia and 18% (7/39) with chronic lymphoproliferative disorder of natural killer cells harbor STAT3 mutations when analyzed by quantitative deep amplicon sequencing. Surprisingly, 17% of the STAT3-mutated patients carried multiple STAT3 mutations, which were located in different lymphocyte clones. The size of the mutated clone correlated well with the degree of clonal expansion of the T-cell repertoire analyzed by T-cell receptor beta chain deep sequencing. The analysis of sequential samples suggested that current immunosuppressive therapy is not able to reduce the level of the mutated clone in most cases, thus warranting the search for novel targeted therapies. Our findings imply that the clonal landscape of large granular lymphocytic leukemia is more complex than considered before, and a substantial number of patients have multiple lymphocyte subclones harboring different STAT3 mutations, thus mimicking the situation in acute leukemia.

Failure to undergo activation-induced cell death due to global dysregulation of apoptosis is the pathogenic hallmark of large granular lymphocyte (LGL) leukemia. Consequently, immunosuppressive agents are rational choices for treatment. This first prospective trial in LGL leukemia was a multicenter, phase 2 clinical trial evaluating methotrexate at 10 mg/m2 orally weekly as initial therapy (Step 1). Patients failing methotrexate were eligible for treatment with cyclophosphamide at 100 mg orally daily (Step 2). The overall response in Step 1 was 38% with 95% confidence interval (CI): 26%, 53%. The overall response in Step 2 was 64% with 95% CI: 35%, 87%. The median overall survival for patients with anemia was 69 months with a 95% CI lower bound of 46 months and an upper bound not yet reached. The median overall survival for patients with neutropenia has not been reached 13 years from study activation. Serum biomarker studies confirmed the inflammatory milieu of LGL but were not a priori predictive of response. We identify a gene expression signature that correlates with response and may be STAT3 mutation driven. Immunosuppressive therapies have efficacy in LGL leukemia. Gene signature and mutational profiling may be an effective tool in determining whether methotrexate is appropriate therapy.

Acute myeloid leukemia (AML) is one of the deadliest leukemias for which there is an urgent and unmet need for the development of novel treatment strategies. Multiple drug resistance mechanisms mediate poor drug response and relapse in patients, and a selective Mcl-1 inhibitor has been speculated to be a promising agent in the treatment of AML. Here, we describe that maritoclax, a small molecule Mcl-1 inhibitor, induces Mcl-1 proteasomal degradation without transcriptional downregulation. Maritoclax killed AML cell lines and primary cells with elevated Mcl-1 levels through selective Mcl-1 downregulation, and synergized with ABT-737 to overcome Mcl-1-mediated ABT-737 resistance. Maritoclax was more effective than daunorubicin at inducing leukemic cell death when co-cultured with HS-5 bone marrow stroma cells, while being less toxic than daunorubicin against HS-5 stroma cells, primary mouse bone marrow cells, and hematopoietic progenitor cells. Moreover, maritoclax administration at 20 mg/kg/d intraperitoneally caused significant U937 tumor shrinkage, as well as 36% tumors remission rate in athymic nude mice, without apparent toxicity to healthy tissue or circulating blood cells. In summary, our studies suggest that maritoclax belongs to a novel class of Mcl-1 inhibitors that has the potential to be developed for the treatment of AML.

Large granular lymphocyte (LGL) leukemia is an indolent lymphoproliferative malignancy which dysregulates humoral immunity and underlies the myriad autoimmune phenomena. We describe a 62-year-old woman with Felty's syndrome who developed a severe bleeding diathesis. Laboratory evaluation demonstrated acquired inhibitors to both factor VIII (FVIII) and fibrinogen, likely secondary to T-cell LGL leukemia. After a complicated course, the patient's inhibitors were extinguished with rituximab and high-dose corticosteroids. Bleeding was controlled with alternating FEIBA (factor eight inhibitor bypassing activity) and recombinant activated FVII. This report reviews the literature comparing the efficacy of various treatment modalities for both disorders. To our knowledge, this is the first reported case of a patient with LGL leukemia acquiring an inhibitor to FVIII or fibrinogen.

The retroviral oncoprotein Tax from human T-cell leukemia virus type 1 (HTLV-1), an etiological factor that causes adult T-cell leukemia and lymphoma, has a crucial role in initiating T-lymphocyte transformation by inducing oncogenic signaling activation. We here report that Tax is a determining factor for dysregulation of autophagy in HTLV-1-transformed T cells and Tax-immortalized CD4 memory T cells. Tax facilitated autophagic process by activating inhibitor of κB (IκB) kinase (IKK) complex, which subsequently recruited an autophagy molecular complex containing Beclin1 and Bif-1 to the lipid raft microdomains. Tax engaged a crosstalk between IKK complex and autophagic molecule complex by directly interacting with both complexes, promoting assembly of LC3+ autophagosomes. Moreover, expression of lipid raft-targeted Bif-1 or Beclin1 was sufficient to induce formation of LC3+ autophagosomes, suggesting that Tax recruitment of autophagic molecules to lipid rafts is a dominant strategy to deregulate autophagy in the context of HTLV-1 transformation of T cells. Furthermore, depletion of autophagy molecules such as Beclin1 and PI3 kinase class III resulted in impaired growth of HTLV-1-transformed T cells, indicating a critical role of Tax-deregulated autophagy in promoting survival and transformation of virally infected T cells.

Research on immigration and crime has only recently started to consider potential heterogeneity in longitudinal patterns of immigrant offending. Guided by segmented assimilation and life course criminology frameworks, this article advances prior research on the immigration-crime nexus in three ways: using a large sample of high-risk adjudicated youth containing first and second generation immigrants; examining longitudinal trajectories of official and self-reported offending; and merging segmented assimilation and life course theories to distinguish between offending patterns. Data come from the Pathways to Desistance study containing detailed offending and socio-demographic background information on 1,354 adolescents (13.6 % female; n = 1,061 native-born; n = 210 second generation immigrants; n = 83 first generation immigrants) as they transition to young adulthood (aged 14-17 at baseline). Over 84 months we observe whether patterns of offending, and the correlates that may distinguish them, operate differently across immigrant generations. Collectively, this study offers the first investigation of whether immigrants, conditioned on being adjudicated, are characterized by persistent offending. Results show that first generation immigrants are less likely to be involved in serious offending and to evidence persistence in offending, and appear to be on a path toward desistance much more quickly than their peers. Further, assimilation and neighborhood disadvantage operate in unique ways across generational status and relate to different offending styles. The findings show that the risk for persistent offending is greatest among those with high levels of assimilation who reside in disadvantaged contexts, particularly among the second generation youth in the sample.